Early release, published at www.cmaj.ca on July 9, 2020. Subject to revision.

RESEARCH Holder pasteurization of donated human milk is effective in inactivating SARS-CoV-2

Sharon Unger MD, Natasha Christie-Holmes PhD, Furkan Guvenc HBSc, Patrick Budylowski HBSc, Samira Mubareka MD, Scott D. Gray-Owen PhD, Deborah L. O’Connor PhD RD

n Cite as: CMAJ 2020. doi: 10.1503/cmaj.201309; early-released July 9, 2020

ABSTRACT

BACKGROUND: Provision of pasteurized ture infectivity dose per mL). We pasteur- using the Holder method. In the SARS- donor human milk, as a bridge to moth- ized samples using the Holder method or CoV-2–spiked milk samples that were er’s own milk, is the standard of care for held them at room temperature for not pasteurized but were kept at room very low-birth-weight infants in hospi- 30 minutes and plated serial dilutions temperature for 30 minutes, we tal. The aim of this research was to con- on Vero E6 cells for 5 days. We included observed a reduction in infectious viral firm that Holder pasteurization (62.5°C comparative controls in the study using titre of about 1 log. for 30 min) would be sufficient to inacti- milk samples from the same donors vate severe acute respiratory syndrome without addition of virus (pasteurized INTERPRETATION: Pasteurization of coronavirus 2 (SARS-CoV-2) in donated and unpasteurized) as well as replicates human milk by the Holder method (62.5°C human milk samples. of Vero E6 cells directly inoculated with for 30 min) inactivates SARS-CoV-2. Thus, SARS-CoV-2. We reported cytopathic in the event that donated human milk

METHODS: We spiked frozen milk sam- effects as TCID50/mL. contains SARS-CoV-2 by transmission ples from 10 donors to the Rogers Hixon through the mammary gland or by con- Ontario Human Milk Bank with SARS- RESULTS: We detected no cytopathic tamination, this method of pasteurization CoV-2 to achieve a final concentration of activity in any of the SARS-CoV-2–spiked renders milk safe for consumption and 7 1 × 10 TCID50/mL (50% of the tissue cul- milk samples that had been pasteurized handling by care providers.

other’s milk is the optimal source of nutrition for infants response to maternal illness and inflammation.3 Other routes of trans- and contains a myriad of bioactive and immunomodula- mission include contamination from respiratory droplets, skin, breast tory factors, including cytokines, lactoferrin, oligosaccha- pumps and milk containers. Milk banks affiliated with the Human Milk Mrides and secretory immunoglobulins, which help orchestrate Banking Association of North America (HMBANA) and the European immune system development and provide first-line defence Milk Bank Association (EMBA) pasteurize milk using the Holder against respiratory tract and gastrointestinal tract infection.1–5 For method (62.5°C for 30 min) before dispensing for use; the Holder vulnerable infants, such as very low-birth-weight (born < 1500 g) method is effective in inactivating the aforementioned viruses.13,14 infants, use of mother’s milk is associated with a shorter hospital Very little is known of the prevalence of severe acute respira- stay and reduces their risk of sepsis and necrotizing enterocolitis, a tory syndrome coronavirus 2 (SARS-CoV-2) in human milk, or its severe bowel emergency.6–9 It is the standard of care in Canada to infectivity; however, the virus has been detected in human milk provide very low-birth-weight infants in hospital with pasteurized by reverse transcription polymerase chain reaction (RT-PCR) donor human milk until their mother’s supply is established.10 testing.15–18 Mothers donating milk are verbally screened for Past global epidemics, such as HIV/AIDS, have had devastating symptoms associated with coronavirus disease 2019 (COVID-19) effects on donor human milk banking because of perceived risks. In at HMBANA-affiliated milk banks, but direct assessment for the 1980s, with the knowledge that HIV could be transmitted into SARS-CoV-2 by nasopharyngeal swabs and RT-PCR testing is not human milk, 22 of the 23 Canadian donor human milk banks performed. Although there is no direct evidence showing that closed.11 Several viruses, in addition to HIV, can be transmitted Holder pasteurization inactivates SARS-CoV-2 in human milk, through human milk, including hepatitis, cytomegalovirus and this virus is known to be heat sensitive.19 The aim of this research human T-cell lymphotropic virus type 1.12 Some viruses may be was to confirm that Holder pasteurization would be sufficient to secreted into milk by paracellular passage as tight junctions open in inactivate SARS-CoV-2 in donated human milk samples.

© 2020 Joule Inc. or its licensors CMAJ 1 Methods undiluted milk-free samples of 160 μL of viral stock solution and 840 DMEM (SARS-CoV-2 alone). Negative controls consisted of Study design unpasteurized and pasteurized human milk with no viral inocu- The Rogers Hixon Ontario Human Milk Bank in Toronto, Canada, lum (mock infection). is a provincial milk bank that follows guidelines established by HMBANA, whereby donors are screened by health and lifestyle Virologic analysis interview and serology, and counselled about safe procedures We observed cytopathic effects at 5 days after infection and RESEARCH

for expression, handling and storage of human milk. Once reported them as TCID50/mL. We calculated the viral titres using donors have collected a minimum volume of milk at home, the the Spearman–Karber method.25,26 At 5 days after infection, we milk is shipped frozen to the milk bank by express priority cour­ passaged supernatants onto fresh Vero E6 monolayers in flat- ier. We chose 1 container of frozen human milk (approximately bottom 96-well plates and refreshed the original monolayers 150 mL) at random from shipments received from each of with 200 μL of DMEM with 2% fetal bovine serum. We monitored 10 donors. The number of samples included align with previous these for 14 days for any signs of emergence of breakthrough investigations of viral inactivation in human bodily fluids, where cytopathic effects not evident in the initial 5 days of culture. The 20–22 it is common to pool samples before spiking with virus. We limit of detection for the TCID50 assay was 20 TCID50/mL. specifically avoided pooling in this study because of the known variability in human milk composition. After all identifiers had Ethics approval been removed from milk containers, they were transported fro- Donors to the Rogers Hixon Ontario Human Milk Bank provide zen to the Combined Containment Level 3 Unit at the University written informed consent that their milk may be used for quality of Toronto, where we completed all experiments. control and research purposes. We obtained human research We thawed the milk samples on ice, homogenized them and ethics approval from Sinai Health and the University of Toronto. individually spiked two 840 μL aliquots of milk from each woman with 160 μL of SARS-CoV-2 SB2 passage 3 (titre = 6.29 × 107 50% Results

of the tissue culture infectivity dose [TCID50] per mL) to achieve a 7 concentration of 1 × 10 TCID50 in each 1 mL of milk-containing Cytopathic effects were identical at 3 and 5 days after infection solution.23 One spiked milk sample from each mother was and are presented in Table 1. We detected no cytopathic activity allowed to sit at room temperature for 30 minutes (unpasteur- in any of the SARS-CoV-2–spiked milk samples that had been ized milk). We pasteurized the second spiked milk sample from pasteurized using the Holder method (62.5°C for 30 min), even each mother in a water bath by warming the milk to 62.5°C, hold- after the passaging of inoculum and subsequent observation for ing for 30 minutes and then cooling in ice to mimic HMBANA and 14 days. Of note, in the positive control samples (SARS-CoV-2 EMBA guidelines.13,14 We processed individual aliquots of milk in hard plastic microfuge tubes of composition similar to contain- Table 1: Stability of SARS-CoV-2 in donated human milk ers normally used during Holder pasteurization at the milk bank. with and without Holder pasteurization (TCID50/mL)* Not unexpectedly, given the complex carbohydrate, lipid and immune factor content of human milk, we found undiluted Unpasteurized human milk to be cytotoxic to Vero E6 cells, even without SARS- (room temperature Pasteurized CoV-2. Hence, we diluted all samples 1:100 in serum-free Sample for 30 min) (62.5°C for 30 min) ­Dulbecco’s Modified Eagle’s Medium (DMEM), the medium used A 2.0 × 105 Undetected to maintain cultures of Vero E6 cells, before conducting the B 6.3 × 104 Undetected experimental procedures described below. Although the dilution 5 of the treated samples does result in a 1-log decrease in sensitiv- C 6.3 × 10 Undetected ity of viable virus detection in the subsequent titration assay, the D 6.3 × 106 Undetected high input titre of SARS-CoV-2 used to spike the milk samples E 6.3 × 105 Undetected balances the dilution factor such that a 6-log reduction in viable F 2.0 × 105 Undetected virus would still be quantifiable. G 6.3 × 105 Undetected We used spiked milk samples to determine viral titres in sam- H 6.3 × 105 Undetected ples from all treatment conditions, as previously described.23,24 5 Briefly, we prepared 6 serial 10-fold dilutions of each SARS- I 6.3 × 10 Undetected CoV-2 milk solution (inoculum) and applied 50 μL of each to J 6.3 × 105 Undetected monolayers of Vero E6 cells with DMEM (0.2 × 106 cells/mL) in SARS-CoV-2 alone 6.3 × 106 6.3 × 103 flat-bottom 96-well plates. We incubated the plates at 37°C and (positive control)

5% CO2 for 1 hour, with gentle shaking every 15 minutes to pro- Mock infection Undetected Undetected mote uniform distribution of the inoculum across the wells. (negative control)

After 1 hour, we removed the inoculum and then reconstituted Note: SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2, TCID50 = 50% of the plate with 200 μL of DMEM with 2% fetal bovine serum, and the tissue culture infectivity dose. *TCID /mL calculations defined by duplicate dilution series of indicated samples. allowed it to progress for 5 days. Positive controls consisted of 50

2 CMAJ alone), this heat treatment did not completely inactivate the Limitations virus. In the SARS-CoV-2–spiked milk samples that were not pas- We studied only 10 samples of milk. However, our ability to study RESEARCH teurized but were kept at room temperature for 30 minutes, we more was limited because of the complexity of ensuring safety observed a reduction of approximately 1 log in comparison with using the SARS-CoV-2 virus, and this sample size is larger than virus spiked into DMEM alone, although we observed variability has been used in similar studies of other viruses.20–22 between milk donors (range of reduction 2 log to none). The 6 6 mean ± standard deviation TCID50/mL was 1.05 × 10 ± 1.86 × 10 . Conclusion Pasteurization of human milk by the Holder method (62.5°C for Interpretation 30 min) inactivates SARS-CoV-2. In the event that a woman who has COVID-19 donates human milk that contains SARS-CoV-2, Very few milk samples from women positive for COVID-19 have whether by transmission through the mammary gland or by con- been tested for SARS-CoV-2. Of the few available cases reported in tamination through respiratory droplets, skin, breast pumps and the literature, there are now at least 3 reports of the presence of milk containers, this method of pasteurization renders milk safe SARS-CoV-2 nucleic acid in human milk, although none of these for consumption. Furthermore, previously frozen, thawed human have measured the viability of the virus in these samples. The milk appears to contain sufficient antiviral activity to partially World Health Organization recommends that human donor milk reduce the infectivity of SARS-CoV-2 in human milk. be fed to low-birth-weight infants when there is an insufficient volume of mother’s milk.27 Human milk banking is growing rap- References idly internationally, with more than 650 milk banks globally that 1. Goldman A, Goldblum R. Chapter 9 — Defense agents in milk: A. Defense agents rely on the Holder method to ensure the safety of donor milk.28 in human milk. In: Jensen R, editor. Handbook of Milk Composition: A Volume in Food Science and Technology. San Diego (CA): Academic Press; 1995:727-45. Although this technique is assumed to result in inactivation of SARS-CoV-2, it is important to confirm this in a human milk 2. Goldman AS. Future research in the immune system of human milk. J Pediatr 2019;206:274-9. matrix, for the safety of milk bank staff, caregivers and recipients 3. Hurley WL, Theil PK. Perspectives on immunoglobulins in colostrum and milk. of human donor milk. In this study, pasteurization of human milk Nutrients 2011;3:442-74. spiked with SARS-CoV-2 using the Holder method (62.5°C for 4. Maertens K, De Schutter S, Braeckman T, et al. Breastfeeding after maternal 30 min) resulted in complete viral inactivation, as measured by immunisation during pregnancy: providing immunological protection to the newborn: a review. Vaccine 2014;32:1786-92. TCID50/mL. The high viral titre used to spike samples in these experiments enabled us to confirm a 106 reduction. The impact 5. Victora CG, Bahl R, Barros AJ, et al.; Lancet Breastfeeding Series Group. Breast- feeding in the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet of pasteurization on coronaviruses in a human milk matrix has 2016;387:475-90. 29 not previously been reported in the literature. 6. O’Connor DL, Gibbins S, Kiss A, et al.; GTA DoMINO Feeding Group. Effect of supple- The results are in keeping with evidence of coronavirus inacti- mental donor human milk compared with preterm formula on neurodevelopment vation in other matrices, including culture media and plasma of very low-birth-weight infants at 18 months: a randomized clinical trial. JAMA 2016;316:1897-905. using a variety of pasteurization protocols. The virus causing 7. O’Connor DL, Jacobs J, Hall R, et al. Growth and development of premature severe acute respiratory syndrome, SARS-CoV, has been shown infants fed predominantly human milk, predominantly premature infant formula, to be completely inactivated with temperatures as low as 56°C or a combination of human milk and premature formula. J Pediatr Gastroenterol for 20 minutes, as well as at higher temperatures, such as 70°C Nutr 2003;37:437-46. for 5 minutes.30–33 The virus causing Middle East respiratory syn- 8. Patel AL, Johnson TJ, Engstrom JL, et al. 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This finding suggested to us that the biological 13. HMBANA. Guidelines for the establishment and operation of a donor human milk matrix in which the virus resides needs to be considered when bank. 10th ed. Fort Worth (TX): Human Milk Banking Association of North America (HMBANA); 2018. assessing effective inactivation conditions. 14. Moro GE, Billeaud C, Rachel B, et al. Processing of donor human milk: update Interestingly, we observed some reduction in the cytopathic and recommendations from the European Milk Bank Association (EMBA). Front effects of SARS-CoV-2 in milk samples that were not heat treated but Pediatr 2019;7:49. held at room temperature for 30 minutes. This is very likely a result 15. Groß R, Conzelmann C, Müller J, et al. Detection of SARS-CoV-2 in human of the multitude of immune components found in human milk — breastmilk. Lancet 2020;395:1757-8. including secretory 1gA antibodies, lactoferrin, lactadherin, mucins 16. Kirtsman M, Diambomba Y, Poutanen SM, et al. 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Competing interests: Deborah O’Connor serves as the Chair of the lectual content, gave final approval of the version to be published and Advisory Board (unpaid) and Sharon Unger serves as the Medical agreed to be accountable for all aspects of the work. Sharon Unger and ­Director (paid) of the Rogers Hixon Ontario Human Milk Bank. No Natasha Christie-Holmes are co-first authors. other competing interests were declared. Funding: This research was funded by the Canadian Institutes of This article has been peer reviewed. Health Research (FDN no 143233). Indirect support was also received from the University of Toronto and the Temerty Foundation to support Affiliations: Rogers Hixon Ontario Human Milk Bank (Unger, O’Connor); enhanced capacity and operations of the Toronto Combined Contain- Department of Paediatrics, Sinai Health (Unger); Combined Contain- ment Level 3 Facility during the COVID-19 pandemic. The sources of ment Level 3 Unit (Christie-Holmes, Guvenc, Budylowski, Gray-Owen), support had no role in the design or conduct of this review, data inter- Departments of Molecular Genetics (Guvenc, Gray-Owen), Laboratory pretation or writing of the manuscript. Medicine and Pathobiology (Mubareka), and Nutritional Sciences (O’Connor), and the Institute of Medical Sciences (Budylowski), University Data sharing: Requests for original data should be made to Dr. Sharon of Toronto; Sunnybrook Research Institute (Mubareka), Toronto, Ont. Unger, at [email protected] Contributors: All of the authors contributed to the conception and design Accepted: June 30, 2020 of the work, and the acquisition, analysis, and interpretation of data. Correspondence to: Sharon Unger, [email protected] Sharon Unger, Natasha Christie-Holmes and Deborah O’Connor drafted the manuscript. All of the authors revised it critically for important intel-

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